Tissue culturing of mammalian cells has become a preferred technique for scientists to study various aspects of cancer, including its etiology and its treatment. A convenient form of tissue culturing is known as two-dimensional monolayer cell culturing. In this technique, cells admixed with appropriate life-sustaining media are placed in a specially-treated plastic petri dish or flask. The cells adhere to the bottom surface of the container, assuming a characteristic flattened pattern during spreading, and replicate on that surface as a single layer, called a monolayer. The media remains on top of the flat layer of cells and is changed periodically to provide the growing cells with essential nutrients. The container wall surface area determines the number of cells that can be effectively cultured. When it is desired to split the cultures, an enzyme such as trypsin is utilized to destroy the anchorage of the cells to the dish so that subcultures can be made. While the cells are in culture, various agents can be applied to the media in the plates and the effect on the cells observed. For example, suspected carcinogens can be added to individual cultures of non-cancerous cells to ascertain if the carcinogen causes the cells to exhibit the growth pattern characteristic of cancerous cells. Tissue culture offers an effective screening tool that increases the number of agents that can be rapidly screened as compared to using animals for the same purpose. With regard to potential treatment of cancerous disease, tissue culture may be used to determine if experimental drugs or antibodies would be effective in destroying cancerous cells. Tissue culture can also be used to attempt to determine whether particular antibodies might bind to cancer cells in order to provide for targeting of particular cells with drugs conjugated to such antibodies.
Even though two-dimensional monolayer tissue culture has provided great benefits to scientists and clinicians, it suffers from a lingering disadvantage as well. Tumors do not grow two-dimensionally in the body, and therefore, monolayer cultures of tumor cells cannot reflect their true in vivo three-dimensional growth architecture. In addition, monolayer cultures reflect a homogeneous cell population in which every cell is exactly like every other cell in culture. This is not the case for solid tumors, which are vascularized within the host and most often exhibit a heterogenous cell population believed to result from cell differentiation induced by differences in biochemical environment such as hormones, growth factors, oxygen tension, and catabolic waste products between blood vessels and the tumor core. The solid tumor has a population of dynamic cells, meaning that they may be constantly changing in response to their environment. Cells may exhibit different morphological, biochemical and histological properties.
In an attempt to more closely model the in vivo tumor, three-dimensional tissue culture has been developed. One technique of three-dimensional tissue culturing is the spinner flask technique. W. Mueller-Klieser, "Multicellular Spheroids," J Cancer Res Clin Oncol 13:101-122 (1986). Another technique is the liquid-overlay technique. J. M. Yuhas, et al., "A simplified method for production and growth of multicellular tumor spheroids," Cancer Res 37:3639-3643 (1977). While three-dimensional growth can be achieved by these techniques, it may be limited as to size and development. Another three-dimensional technique is that developed by the National Aeronautic And Space Agency ("NASA") which is a rotating culture vessel specifically engineered to randomize the gravity vector by rotating a fluid-filled culture vessel about a horizontal axis while suspending cells and cell aggregates with minimum fluid shear. These devices have been described in U.S. Pat. Nos. 5,153,131; 5,153,132; 5,153,133; 5,155,034; and 5,155,035. A commercial device for three-dimensional cell culturing is known as the High Aspect Rotating Vessel (HARV) and is manufactured by Synthecon, Inc. (Friendswood, Tex.). The HARV vessel, or bioreactor, rotates cells and medium in a disk-shaped 50 ml growth chamber about a horizontal axis with zero head space, resulting in low fluid shear conditions. Adequate gas exchange is maintained across a siliconized rubber membrane which lines one face of the chamber.
Despite the advent of three-dimensional cell culturing techniques, many obstacles remain in the diagnosis and treatment of cancer. The techniques are available as tools, but these tools have not been implemented in the prior art to provide a diagnostic tool for urological cancers, such as prostate and bladder cancer.
Prostate cancer is the most common non-cutaneous malignant disease among American males, and presumably is also a world-wide health problem. The incidence of prostate cancer increases more rapidly with age than any other type of cancer. Prostate cancer often causes death while remaining undiagnosed. A. W. Meikle, et al., "Epidemiology of prostate cancer," in Early Detection and Treatment of Localized Carcinoma of the Prostate. J. A. Smith (ed.) Saunders, Phila., pp. 709-718 (1990).
At present, prostate cancer, or adenocarcinoma (defined as a malignant neoplasm of epithelial cells in glandular or gland-like pattern), has been diagnosed or predicted from histologic grading, staging, tumor volume and a multi-parameter predictive factor score (PFS). D. F. Gleason, "Prediction of Prognosis for Prostatic Adenocarcinoma by Combined Histological Grading and Clinical Staging," J Urol 111:58-64 (1974); D. F. Gleason, "Histological Grading of Prostate Carcinoma," in Contemporary Issues in Surgical Pathology of the Prostate. D. G. Bostwick (ed.), Churchill Livingstone, Edinburgh, pp. 83-93 (1990); Partin et al., "Use of nuclear morphometry, Gleason histologic scoring, clinical stage, and age to predict disease-free survival among patients with prostate cancer," Cancer 70:161-168 (1992); Partin, et al., "A comparison of nuclear morphometry and Gleason grade as a predictor of prognosis in stage A2 prostate cancer: a critical analysis," J Urol 142:1254-1258 (1989). The success of these predictive methods depends heavily on the subjective and selective objective interpretative skills of a clinician.
To eliminate the subjective nature of histologic analysis described above, the art has suggested that biomarkers be observed. The term "biomarker" can be generally defined as a genetically determined product expressed as antigenic proteins, glycoproteins or other biomolecules detectable by antibody probes. These include growth factors, hormones, receptors, oncogenes, cytoskeletal and nucleoskeletal proteins, tumor suppressor gene products, differentiation molecules or organ-restricted neoantigens.
Nuclear morphometric measurements, or "shape descriptors" have prognostic value in determining the clinical outcome of prostatic malignancies. Pressman, N.J., "Markovian analysis of cervical cell images," J Histochem Cytochem 24:138-144 (1976). Nuclear shape is a function of the interaction of nuclear proteins and cytoplasmic structural determinants. Diamond et al demonstrated that nuclear roundness could distinguish prostatic tumors with high metastatic potential from those that would eventuate as more indolent tumors. Diamond et al., "Computerized image analysis of nuclear shape as a prognostic factor for prostatic cancer," The Prostate 3:321-332 (1982). Partin et al utilized variance of nuclear roundness incorporating it into the multi-parameter predictive factor score (PFS) mentioned above. Partin et al., Cancer 70:161-168 (1992). Three-dimensionality provides a micro-milieu in which these determinants can more realistically recreate an in vivo-like interdependence.
One biomarker that has been followed in the art to determine the progression of disease is DNA ploidy analysis. The term "ploidy" refers to the state of the cell nucleus with respect to the number of genomes it contains. A genome is the complete set of chromosomes derived from one parent, or haploid number, so in a normal non-gametic cell, the ploidy is diploid. Cancer cells may be diploid, tetraploid, or aneuploid (having an abnormal number of chromosomes, not an exact multiple of the haploid number). DNA ploidy analysis has been found to be of prognostic significance in selected prostate cancers. J. M. Peters, et al., "Prognostic significance of the Nuclear DNA Content in Localized Prostatic Adenocarcinoma," Anal Quant Cytol Histol 12:359-365 (1990); R. W deVere White, et al., "Prognosis in Disseminated Prostate Cancer As Related to Tumor Ploidy and Differentiation," World J Urol 8:47-50 (1990); R. A. Stevenson, et al., "Flow Cytometry of Prostate Cancer: Relationship of DNA Content to Survival," Cancer Res 47:2504-2509 (1987).
While a correlation between DNA ploidy patterns and prognostics has been established, data have also suggested that the ploidy patterns may vary between the peripheral zone and the central zone of tumor specimens. However, three-dimensionally cultured cells and tissue-like aggregates have not been used for the purpose of patient-specific diagnosis, monitoring, and therapeutics, i.e., identification of patterns of biomarker panel expressions and the identification of new biomarkers as neoantigens within individual clinical patients.
Another positive marker of cellular differentiation in the malignant or premalignant state has been reported to be the increased level of f-actin, a polymerized form of actin, a ubiquitous cytoskeletal protein in eukaryotic cells associated with cellular motility and morphology, intracellular transport, secretion and cell division.
Cancer of the prostate may also be regulated by cellular oncogenes or tumor suppressor genes. In an isogenic male murine host, retrovirally introduced myc and ras oncogenes induced hyperplastic and dysplastic pathologies, respectively, in the reconstituted fetal urogenital sinus model. Myc+ras in combination induced carcinomas. Thompson et al., "Multistage carcinogenesis induced by ras and myc oncogenes in a reconstituted organ," Cell 56:917-930 (1989). The same mouse model noted a strong association of Transforming Growth Factors (TGF), i.e., TGF-.beta.1 and TGF-.beta.3 in prostatic tumor progression. Thompson, T. C., "Growth factors and oncogenes in prostate cancer," Cancer Cells 2:345-354 (1990). The c-erbB2 (HER-2/neu) oncogene product has been detected immunohistochemically at significant levels in human prostatic carcinomas. Kuhn et al., "Expression of the c-erbB2 (HER-2/neu) oncoprotein in human prostatic carcinoma," J Urol 150:1427-1433 (1993); Ware et al., "Immunohistochemical detection of c-erbB2 protein in human benign and neoplastic prostate," Human Pathology 22:254-258 (1991).
High levels of the ras oncogene have been detected in malignant prostate and benign prostatic hypertrophy (BPH) specimens. Higher levels of c-myc messenger RNAs have been reported in human adenocarcinoma than in BPH. Abnormal expression of p53 tumor suppressor gene has been demonstrated in at least two prostate carcinoma cell lines. The Rb, or retinoblastoma gene product, expression may be impaired or defective in human prostate carcinomas as are other markers of metastatic disease.
Another biomarker of metastatic prostatic disease is Prostatic Specific Antigen (PSA). This is a serum-based biomarker and is generally recognized as a good indicator of prognosis, but a significant number of false positive radioimmunoassays will occur in cases of BPH. W. M. Lineham, et al., "Metastatic models and molecular genetics of prostate cancer," J Natl Cancer Inst 84:914-915 (1992).
Other biomarkers that have been elucidated are TURP-27 (G. L. Wright, et al., "A novel prostate carcinoma-associated glycoprotein complex recognized by monoclonal antibody TURP-27," Int J Cancer 47:717-725 (1991); G. L. Wright, et al., "Immunohistochemical evaluation of the expression of prostate tumor-associated markers in the nude mouse human prostate carcinoma heterotransplant line PC-82, PC-EW, and PC-EG," The Prostate 17:301-316 (1990)), PSP-19 (G. L. Wright, et al., "Generation and characterization of monoclonal antibodies to prostate secretory protein (PSP)," Int J Cancer 46:39-49 (1990); G. L. Wright, et al., The Prostate 17:301-316 (1990)) and PD-41 (M. L. Beckett, et al., "Monoclonal antibody PD-41 recognizes an antigen restricted to prostate adenocarcinomas," Cancer Res 51:1326-1333 (1991); G. L. Wright, et al., The Prostate 17:301-316 (1990)). Another marker binds to monoclonal antibody MCA-R1 but does not bind to BPH-associated tissue. A. Tjota, et al., "Murine monoclonal antibodies reactive with a variety of androgen independent Dunning rat prostate adenocarcinoma sublines are also reactive with human prostate adenocarcinoma," J Urol 146:205-212 (1991).
Superficial transitional cell bladder cancer is another form of urological carcinoma which is commonly diagnosed and treated in the early stages of tumor development and is potentially curable. However, recurrence of the bladder cancer occurs in over 50% of patients after successful initial treatment, and in about 30% of the patients, recurrence progresses to infiltrating cancer. Because of the variability in clinical behavior in individuals, patient management requires a constant monitoring of the bladder. Clinical methods of predicting recurrence and disease progression include tumor staging, tumor size and patient's previous recurrence rate. Biological predictors have been described for testing both tumor specimens and exfoliated cells which include DNA ploidy, oncogene or tumor suppressor gene activation or inactivation (e.g., H-ras, c-erbB2, Rb, p53), rate of epidermal growth factor expression (e.g., AMFr, autocrine motility factor), and the reaction of specific monoclonal antibodies to antigens selectively expressed on bladder tumors. For example, monoclonal antibodies to the highly restricted tumor-associated M-344 and 19a211 antigens having been characterized as biomarkers of high specificity for papillary superficial bladder tumors and carcinoma in situ, whereas T43 and T138 antigen expression is associated with progression to invasive cancer. Cordon-Cardo, et al., "Immunopathologic analysis of human urinary bladder cancer. Characterization of two new antigens associated with low-grade superficial bladder tumors," Am J of Pathology 140:375-385 (1992); Fradet, Yves, "Markers of prognosis in superficial bladder cancer," Seminars in Urology X:28-38 (1992)
It has now been found that three-dimensional culturing techniques can be employed to induce selective differentiation of phenotypic, nuclear morphometric parameters, motility and genotypic markers on tumor cells associated with urological cancers, specifically prostate and bladder adenocarcinoma. The use of three-dimensional culturing techniques is superior to both the use of two-dimensional techniques and the study of biopsied tissue specimens because the three-dimensional culture is believed to more closely resemble the in vivo dynamic tumor tissue. The method of this invention is useful for diagnostic and therapeutic applications.